ANALYSIS OF HYPERMUTATION IN IMMUNOGLOBULIN HEAVY-CHAIN PASSENGER TRANSGENES

Somatic hypermutation of immunogloblin (Ig) genes plays a critical rol e in the maturation of the human antibody response. The molecular basi s of this important process is, however, unknown. To identify cis-acti ng sequences that initiate and target hypermutation, we have made thre e minitransgenes containing different portions of an Ig heavy chain (I gH) locus. Each transgene is a passenger, bearing a nonsense mutation preventing its translation: thus. transgene mutations reflect the endo genous mutational process and are not subject to affinity selection. T o study transgenes after their circulation through the compartment ass ociated with hypermutation in vivo, we rescued B cells as hybridomas a fter hyperimmunizing mice with the hapten 4-hydroxy-3-nitrophenyl acet yl (NP). Hybridoma transgene and endogenous variable regions were ampl ified by polymerase chain reaction. subcloned, and sequenced. Endogeno us anti-NP VDJ regions show the expected. at times extensive degree of base substitution. In mice bearing the smallest construct, which incl udes 2.4 kb of 5' IgH sequences. a rearranged VDJ region. the 5' matri x attachment region. and the intron enhancer, one of four evaluable hy bridomas demonstrates two base substitutions in the V segment of one t ransgene copy. The two larger constructs include additional 3' IgH seq uences (an alpha constant region and the 3' enhancer) and hybridomas d erived from mice bearing these larger constructs demonstrate no eviden ce of targeted mutation, despite demonstrable transgene transcription in all hybridomas. In our system, mutation of a rearranged VDJ segment and surrounding promoter/enhancer regions is not increased by the jux taposition of a constant region segment and the IgH 3' enhancer.